1. Field of the Invention
The present invention generally relates to a HA Receptor for Endocytosis, and more particularly, but not by way of limitation, to methods of purifying such HA Receptor for Endocytosis.
2. Brief Description of the Related Art
HA, also referred to herein as hyaluronic acid, or hyaluronan, is an important and often abundant extracellular matrix component of all tissues, in particular cartilage, skin and vitreous humor (Evered and Whelan, The Biology of Hyaluronan, Ciba Fnd. Symposium, 143:1 (1989)). HA plays a key role in development, morphogenesis and differentiation, in cell adhesion and proliferation, and in inflammation and wound healing (Evered and Whelan, The Biology of Hyaluronan, Ciba Fnd. Symposium, 143:1 (1989); Toole, J. Intern. Med. 242:35 (1997); Knudson and Knudson, FASEB J. 7:1233 (1993); Laurent and Fraser, FASEB J. 6:2397 (1992)). In humans, the total body turnover of HA is several grams per day (Evered and Whelan, The Biology of Hyaluronan, Ciba Fnd. Symposium, 143:1 (1989)). Although local turnover of HA occurs in avascular tissues, particularly cartilage (Hua et al, J. Cell Sci. 106:365 (1993); Aguiar et al, Exp. Cell Res. 252:292 (1999)), two major clearance systems are responsible for HA degradation and removal in the body (Laurent and Fraser, FASEB J. 6:2397 (1992)). The first is the lymphatic system, which accounts for about 85% of the HA turnover, and the second is in the liver, which accounts for the other approximately 15% of the total body HA turnover.
Throughout the body, HA is continuously synthesized and degraded in almost all tissues. At the same time, chondroitin sulfate and other glycosaminoglycans are also released from the cleavage of proteoglycans, especially aggregating proteoglycans associated with HA. Large native HA molecules (about 107 Da) are partially degraded into large fragments (about 106 Da) that are released from the matrix and enter the lymphatic system, thereafter flowing to lymph nodes.
The lymph nodes completely degrade the majority of HA (about 85%) by currently unknown mechanisms. Neither the responsible cell type, the receptor involved, nor the location in lymph nodes at which HA uptake and degradation occurs has been determined. The remaining HA (about 15%) that escapes degradation in the lymph nodes ultimately flows into the blood at the thoracic duct. Since HA is an exceptionally viscous polysaccharide in solution, it would be deleterious for the blood concentration of HA, even at relatively low molecular weight, to increase. Clearance of this circulating HA and the other glycosaminoglycan degradation fragments, such as chondroitin sulfate, is presumably important for normal health (Evered and Whelan, The Biology of Hyaluronan, Ciba Fnd. Symposium, 143:1 (1989); Laurent and Fraser, FASEB J. 6:2397 (1992)). Elevated serum HA levels are associated with a variety of diseases and pathological conditions such as liver cirrhosis, rheumatoid arthritis, psoriasis, scleroderma, fibromyalgia and some cancers (Yamad et al, Acta Haematol. 99:212 (1998); Lai et al, J. Lab Clin. Med. 131:354 (1998); Yaron et al, J. Rheumatol. 24:2221 (1997)).
Liver endothelial cells (LECs) in vertebrate liver express a very active, recycling endocytic receptor that removes these extracellular matrix-derived fragments of HA and other glycosaminoglycans, including chondroitin sulfate, from the blood (Laurent and Fraser, FASEB J. 6:2397 (1992); DeBleser et al, Gut, 35:1509 (1994); Raja et al, J. Biol. Chem. 263:16661 (1988); McGary et al, Biochem. J. 257:875 (1989); McGary et al, Hepatology, 18:1465 (1993)). ICAM-1, a 90 kDa protein also known as CD54 (Hayflick et al, Immunol. Res. 17:313 (1998)), was previously misidentified as the LEC HA Receptor for Endocytosis (HARE) (Forsberg and Gustafson, Biochim. Biophys. Acta, 1078:12 (1991); McCourt et al, J. Biol. Chem. 269:30081 (1994)). This research attempted to purify the HA receptor without the use of an assay to measure HA-binding activity. The claim that the HA Receptor for Endocytosis had been purified was subsequently acknowledged to be an artifact due to the nonspecific binding of ICAM-1 to the HA affinity resin (McCourt and Gustafson, Int. J. Biochem. Cell Biol. 29:1179 (1997); McCourt et al, Hepatology 30:1276 (1999)). In any case, since ICAM-1 is not a coated pit-targeted endocytic receptor, it is not the true HA receptor in LECs.
In addition to the normal turnover of HA in tissues throughout the body, a wide range of biomedical and clinical applications use exogenous HA that is also removed from the lymphatics or ultimately from the blood and degraded by the LEC HARE. For example, HA is used extensively in eye surgery, in the treatment of joint diseases including osteoarthritis, and is being developed as a drug delivery vehicle. Numerous studies have explored the benefit of HA during wound healing. The exogenous HA introduced in these various applications is naturally degraded by the lymph and LEC systems noted above. Despite the very large endocytic and degradative capacity of the LEC HARE and its importance in removing HA from the blood, prior to the present invention the HARE had not been successfully purified, molecularly cloned or expressed from a cDNA.
In two previous studies, one using a photoaffinity derivative of HA (Yannariello-Brown et al, J. Biol. Chem. 267:20451 (1992)) and the other using a novel ligand blot assay with 125I-HA (Yannariello-Brown et al, Glycobiol. 7:15 (1997)), two specific HA-binding proteins in isolated rat LECs were identified at about 175 kDa and about 300 kDa. However, it was not until the present invention that the role of these two proteins in degradation of HA was determined.